Altered cellular energetics contributes to human disease in general, physiological aging and chronic diseases in particular, including cancer, neurodegeneration, ischemic heart disease and type 2 diabetes. Pathological changes affecting the individual may arise only in specific cells of a tissue, and potentially in a heterogeneous manner. To advance basic research of such conditions a commercial kit will be designed for bioenergetics characterization of single cells or subpopulations of cells in heterogeneous, micro-scale samples, such as primary cell and tissue cultures or freshly isolated tissues. The kit follows a wet-bench / dry-bench approach, that consists of an assay paradigm applicable to typical commercial microscopy systems (wide field, confocal and two-photon fluorescence microscopes) with reagents, media and protocols, plus software specialized for analysis of data collected using the assay paradigm. The characterization will be performed by assaying mitochondrial membrane potential (??M), because in contrast to the commonly used cellular energetic descriptor, cell respiration, ??M can be determined on the single cell level. Assaying ??M has been an invaluable basic research tool, but until recently determination of the absolute magnitude of ??M in intact cells was unattainable in a generalizable manner, and instead qualitative and semi-quantitative assays, prone to data misinterpretation due to a series of biasing factors have been used. We have recently introduced a novel technology that uses a biophysical model-based calibration of ??M in single cells to calculate the absolute magnitude ??M and account for effects of all known biasing factors. This calibration algorithm and its software implementation in Image Analyst MKII by the applicant small business concern (Image Analyst Software) forms a core technology, and development of kit-based applications to measure specific cellular energetic parameters using this core technology are proposed here. A commercial product will be developed that provides simplification and decoding of this technically challenging approach for a wider audience of researchers in biomedical sciences. The combination of an assay kit with robust commercial software will enable dissemination of this technology. In this Phase I STTR project assay protocols will be developed, simplified and benchmarked by using quality control internal to the calibration algorithm and comparison to cellular oxygen consumption and proton production measurements. The software implementation of the calibration algorithm, the Membrane Potential Calibration Wizard, will be hardened and its interactivity will be enhanced. Phase II will aim for the development of the actual kits and optimization of the kits for a wider range of specimens including primary, differentiated stem cell and organotypic cultures besides commonly used cell lines.